The goal of this project is to determine the molecular structure of the adaptive landscapes across which two enzymes evolve. By relating enzyme structure to enzyme function, enzyme function to metabolic flux, and metabolic flux to Darwinian fitness, this work will provide a detailed understanding of the causes of adaptation and constraint in biochemical evolution. Phylogenetic analyses reveal that 3.5 billion years ago an ancient bacterial NAD-dependent isocitrate dehydrogenase evolved the ability to utilize NADP. In contrast, all known isopropylmalate dehydrogenases utilize NAD. Protein engineering has confirmed that only 6 out of 250 amino acid replacements determine which coenzyme is used. With so few sites determining coenzyme usage, all possible genetic intermediates between the two extreme phenotypes can be constructed. Competition between strains of Escherichia coli carrying different mutant alleles will be used to determine fitnesses. Thus, the relations between catalytic efficiency, substrate specificity and fitness will be rigorously determined, enabling the molecular basis of the adaptive shift in coenzyme utilization by isocitrate dehydrogenase (for growth on acetate), and the constraints that force isopropylmalate dehydrogenase to use NAD (enzymes with intermediate phenotypes are less fit) to be understood in terms of adaptive landscapes. By investigating what has, and has not, happened during 4 billion years of molecular evolutionary history will not only enrich our understanding of biochemical adaptation, but may also provide subtle insights into the relations between protein structure and function, ones that might be overlooked by more traditional approaches. Many of these may prove helpful to the rational design of catalysts for industry, and of drugs for medicine.